The major focus of this grant is to identify the molecular mechanisms that regulate chondrocyte maturation during endochondral bone formation. During this process, chondrocytes undergo a differentiation process characterized by hypertrophy and production of a unique protein, collagen X (Col10). Thyroid hormone (T3), bone morphogenetic proteins (BMPs) and retinoic acid (RA, the major active metabolite of vitamin A) exert profound effects on this process. Each of these signaling pathways is essential for endochondral bone formation and cannot compensate for the others;abrogation of any one pathway (e.g. hypothyroidism, which blocks T3 signaling) prevents differentiation, resulting in shortened bones. Regulation of the Col10 gene provides a window into the molecular mechanisms by which T3, RA and BMPs control chondrocyte maturation. During the current funding period, we have shown that the T3, RA and BMP signaling pathways converge on a small distal region of the chick Col10 promoter that contains binding sites for multiple transcription factors, including RA receptors, Runx2, an Ets family member and two DNA-bending factors, Lef1 and AP1. All of these sites play a important roles in both basal and RA/BMP-stimulated transcriptional activity;thus this enhancer may function as an integrator of the various signaling pathways that activate Col10 gene transcription. We have demonstrated a hierarchical network of these effectors, in which T3 acts indirectly on Col10 gene expression by stimulating both retinoid and BMP signaling. In addition, we have shown that RA has both direct and indirect effects, stimulating Col10 transcription directly, but also further stimulating BMP signaling. In this renewal application, we propose the following specific aims: 1) We will test the hypothesis that alterations in binding of transcription factors and comodulators that alter promoter and chromatin architecture are responsible for the dramatic increase in Col10 that occur during chondrocyte maturation in vivo and in response to T3, BMPs and RA in culture;and 2) We will test the hypothesis that T3 activates Col10 transcription through stimulation of BMP and RA signaling and this stimulation is essential for T3 effects. The in vivo experiments will utilize two unique mouse models with targeted inactivation of thyroid hormone receptors. One strain displays skeletal hypothyroidism, resulting in delayed endochondral bone formation, while the other displays skeletal thyrotoxicotis, resulting in accelered endochondral ossification;both result in short stature. These experiments will provide significant mechanistic insights into the problems of short stature due to failure of growth plate differentiation or premature growth plate closure. PUBLIC HEALTH RELEVANCE: Skeletal growth is profoundly affected by thyroid hormones, vitamin A and bone morphogenetic proteins (BMPs). There are millions of people in this country with undiagnosed thyroid disease and many more millions worldwide with vitamin A deficiency;furthermore, vitamin A derivatives and BMPs are in widespread clinical use, and the impact of their use on skeletal growth is not well understood. The proposed experiments will provide significant insights into the roles these growth factors and hormones play in failure of normal skeletal growth, resulting in short stature.